Isoxazole and crotonamide derivatives and their use as pharmaceuticals and diagnostics

ABSTRACT

A compound of the formula ##STR1## where R 1  is the radical of the formula II or III ##STR2## is suitable for the production of a pharmaceutical for the treatment of inflammations, carcinomatous diseases, or autoimmune diseases. A compound of the formula IV ##STR3## is suitable for the production of specific antibodies against a compound of the formula I for the discovery of specific-binding proteins from cell extracts, serum, blood, or synovial fluids, for the purification of proteins, for the modification of microtiter plates, or for the preparation of chromatography material, in particular of affinity chromatography material, and for use in diagnostics.

The invention relates to novel isoxazole and crotonamide derivatives,their preparation and use as pharmaceuticals, and their use as anantigen for the production of antibodies and their use in diagnosis andpurification processes.

BACKGROUND OF THE INVENTION

Isoxazole and crotonamide derivatives having antiinflammatory,immunosuppressant, or antiproliferative action have been disclosed (EP 0013 376; EP 02 217 206; EP 0 527 736). The analytical determination ofthese compounds in animal and human sera is possible with the aid ofconventional chromatographic processes. The disadvantage of thesechromatographic processes is a high outlay in terms of apparatus,complicated sample preparation steps and low sample throughput.

Immunological determination and analysis processes are a rapid andreliable alternative to chromatographic processes. The production ofsuitable antibodies is crucial for carrying out these alternativeprocesses.

SUMMARY OF THE INVENTION

The invention aims, by modification of isoxazole and crotonamidederivatives, to make available compounds which are suitable for antibodyproduction, can be coupled to polymers and can be employed as tracers inradioimmunoassays.

It has been found that compounds of the formula I are suitable forachieving this object where, on the aromatic ring of the aniline moiety,there are one or more functional groups which can be coupled covalentlyto polymers as such or via a spacer function.

The invention therefore relates to compounds of the formula I ##STR4##and/or a physiologically tolerable salt of the compound of the formula Iand/or an optionally stereoisomeric form of the compound of the formulaI, where R¹ is the radical of the formula II or III ##STR5## and R² isa) --O--(CH₂)_(n) --CH═CH₂, in which n is the integer 1, 2, or 3,

b) --O--(CH₂)_(m) --CH₂ -halogen, in which m is the integer 1, 2, or 3and halogen is fluorine, chlorine, bromine, or iodine,

c) the radical of the formula V ##STR6## in which R³ is

1) halogen or

2) --NH₂ and

R⁴ is

1) a hydrogen atom or

2) a radical of an amino acid, or

d) --NH₂.

DETAILED EMBODIMENTS OF THE INVENTION

Preferred compounds of the formula I are those wherein

R¹ is the radical of the formula II or III and

R² is

a) --O--(CH₂)_(m) --CH₂ -halogen, in which m is the integer 2 andhalogen is bromine or iodine,

b) --O--CH₂ --CH═CH₂ or

c) --NH--C(O)--CH(R³)(R⁴), in which R³ is bromine, --NH₂, or chlorineand R⁴ is a hydrogen atom.

Particularly preferred compounds of the formula I are those such as2-cyano-3-hydroxybut-2-enecarboxylic acid (4-allyloxyphenyl)amide,2-cyano-3-hydroxybut-2-enecarboxyiic acid(4-(3-iodopropoxy)phenyl)amide, 2-cyano-3-hydroxybut-2-enecarboxylicacid (4-(2-aminoacetylamino)phenyl)amide, 5-methylisoxazole-4-carboxylicacid (4-(2-aminoacetylamino)phenyl)amide,2-cyano-3-hydroxybut-2-enecarboxylic acid(4-(2-bromoacetylamino)phenyl)amide, or 5-methylisoxazole-4-carboxylicacid (4-(2-bromoacetylamino)phenyl)amide.

The radical R² in formula I can be in the meta-, ortho-, orpara-position relative to the "NH" group on the phenyl ring, preferablyin the para-position.

The compounds of the formula I can optionally be present as opticalisomers, diastereomers, racemates, or as mixtures thereof. The term"amino acid" is understood as meaning the stereoisomeric forms, e.g., Dand L forms, of the following compounds: asparagine, valine, arginine,aspartic acid, glutamine, glutamic acid, tryptophan, β-alanine, lysine,proline, glycine, γ-aminobutyrate, Nε-acetyllysine, Nσ-acetylornithine,Nγ-acetyldiaminobutyrate, Nα-acetyldiaminobutyrate, histidine,isoleucine, ieucine, methionine, phenylalanine, serine, cysteine,threonine, alanine, and tyrosine. L-Amino acids are preferred. The aminoacid residue Gly is particularly preferred.

Amino acid residues are derived from the corresponding amino acids. Thebrief notation for the amino acids follows the generally customarynotation. The radical (R⁴) represents the side chain of the respectiveamino acid.

Suitable physiologically tolerable salts of the compounds of the formulaI are, for example, alkali metal, alkaline earth metal and ammoniumsalts, including those of organic ammonium bases and salts of theprotonated amino acid residues.

The invention also relates to a process for the preparation of acompound of the formula I and/or a physiologically tolerable salt of acompound of the formula I and/or an optionally stereoisomeric form of acompound of the formula I, which comprises:

a) reacting a compound of the formula VI ##STR7## where R⁶ is theradical OH Cl, or Br, with a compound of the formula VII ##STR8##

where R⁷ is

1) --NH₂,

2) an --NH--C(O)--CH₂ --NH-protective group, in which protective groupis an amine protective group, for example Boc,

3) --NH--C(O)--CH₂ --halogen or

4) --OH

to give a compound of the formula I in which R¹ is the radical of theformula II and R² is --NH₂, --NH--C(O)--CH₂ --NH-protective group, --OH,or --NH--C(O)--CH₂ -halogen, or

b) reacting a compound prepared by process a), in which R⁷ is --OH, withan alkyl halide or a dihaloalkane in which the alkyl moiety has 2, 3, or4 carbon atoms to give a corresponding compound of the formula I, or

c) reacting a compound prepared by process a), in which R⁷ is --OH, withan unsaturated alkyl halide in which the alkyl moiety has 3, 4, or 5carbon atoms to give a corresponding compound of the formula I, or

d) reacting a compound prepared by process a), in which R⁷ is --NH₂,with a carboxylic acid halide such as bromoacetyl bromide to give acompound of the formula I in which R² is the radical of the formula V,R³ is halogen and R⁴ is a hydrogen atom, or

e) reacting an aromatic diamine such as p-phenylenediamine with an aminoacid protected on the amino group to give a compound of the formula VIIin which R⁷ is a radical of the formula V, R³ is --NH₂ and R⁴ is aprotected amino acid and then reacting as in process a) to give acorresponding compound of the formula I, or

f) removing the protective group in a compound of the formula I preparedby process a) or e), or

g) converting a compound of the formula I prepared by processes a)through f), where R¹ is the radical of the formula II, into a compoundof the formula I where R¹ is the radical of the formula III, or

h) either isolating the compound of the formula I prepared by processesa) through g) in free form or, in the case of the presence of acidic orbasic groups, optionally converting it into physiologically tolerablesalts.

In the process step according to a), it is possible to convert, forexample, an isoxazole-4-carboxylic acid (R⁶ is --OH) into an acidchloride by methods known from the literature, e.g., by means of thionylchloride or phosphorus oxychloride in an aprotic solvent (e.g., toluene,tetrahydrofuran (THF) or a chlorinated hydrocarbon) and then to react itwith an aromatic amine which is optionally substituted in a suitablemanner with addition of an organic base, e.g., with a tertiary amine(e.g., triethylamine or N-ethylmorpholine), in a dipolar aproticsolvent, for example THF or a chlorinated hydrocarbon. Alternatively,the amide formation can be carried out directly from the carboxylic acidwith addition of a condensing reagent known from peptide chemistry,e.g., dicyclohexylcarbodiimide. The second variant is particularlysuitable for aromatic amines having further amino or alcohol radicals.

In process steps b) or c), the hydroxyl group introduced by means of theaniline moiety is further functionalized by reacting this:

1) with an alkyl halide or a dihaloalkane such as 1,3-diiodopropane withaddition of an organic or inorganic base, e.g., potassium carbonate, togive a correspondingly substituted araliphatic ether, where in thelatter case dimerization can be avoided by an appropriately large excessof alkylating agent or use thereof as a solvent, such that in theproduct an alkyl halide function remains for the further coupling of theproduct to a stationary phase, or

2) reacting with an unsaturated alkyl halide, e.g., an allyl orpropargyl halide, preferably allyl bromide, in a dipolar aproticsolvent, such as tetrahydrofuran (THF), acetone or chlorinatedhydrocarbons under similar conditions to an allyl or propargyl ether,where when using acetone as a solvent and potassium carbonate as a basesimultaneously the ring opening of the isoxazole moiety (radical of theformula II) described under g) occurs.

In process step d), the amino group is reacted with a carboxylic acidhalide, e.g., bromoacetyl bromide, with addition of an organic base suchas a tertiary aromatic amine (e.g., triethylamine or N-ethylmorpholine)in a dipolar aprotic solvent such as THF or a chlorinated hydrocarbon.

In process step e), an aromatic diamine, e.g., p-phenylenediamine, isconverted into the monoamide in a controlled manner using an amino acidprotected on the amino group, e.g., N-Boc-glycine, using a condensingagent, e.g., dicyclohexylcarbodiimide, then the anilide formation iscarried out as described under process step a), and finally the amineprotective group carried along is removed under standard conditionsknown from peptide chemistry (process step f)).

In process step g), a compound prepared under a) through f) is subjectedto a base-induced ring opening of the isoxazole moiety, the reactionpreferably being carried out in an aqueous/organic solvent mixture,e.g., THF/water or ethanol/water, using an excess of an organic orinorganic base, e.g., NaOH, ammonia solution, or potassium carbonate.

Provided the compounds of the general formula I occur indiastereoisomeric or enantiomeric forms and are obtained in the selectedsynthesis as mixtures thereof, separation into the pure stereoisomers iscarried out either by chromatography on an optionally chiral supportmaterial, or provided the racemic compounds of the formula I are capableof salt formation, by fractional crystallization of the diastereomericsalts formed with an optically active base or acid as auxiliary. In amanner which is identical in principle, the racemic compounds of theformula I which contain a basic group such as an amino group can beconverted into the pure enantiomers using optically active acids such as(+)-camphor-10-sulfonic acid, D- and L-tartaric acid, D- and L-lacticacid or (+) and (-)-mandelic acid.

The invention also relates to pharmaceuticals which contain anefficacious amount of at least one compound of the formula I and/or astereoisomeric form of the compound of the formula I and/or aphysiologically tolerable salt of the compound of the formula I, inaddition to pharmaceutically suitable and physiologically tolerableexcipients, additives and/or active compounds and auxiliaries. Thepharmaceuticals according to the invention can be administeredintravenously, parenterally, topically, rectally, or orally.

The pharmaceuticals according to the invention are preferably suitablefor the prophylaxis and/or therapy of carcinomatous diseases,inflammations, and autoimmune diseases.

These include, for example, rheumatic diseases, acute, and chronicinflammations of muscles, joints, or of the gastrointestinal tract,allergic airway diseases, psoriasis, or autoimmune diseases, e.g.,systemic lupus erythematosus ("SLE"), type II diabetes, myastheniagravis, Sjogren syndrome, dermatomyositis, sclerodermatitis, or multiplesclerosis ("MS"). The carcinomatous diseases include, for example, lungcancer, leukemia, Kaposi's sarcoma, ovarian cancer, sarcoma, meningioma,intestinal cancer, cancer of the lymph nodes, brain tumors, breastcancer, stomach cancer, cancer of the pancreas, cancer of the prostate,or skin cancer.

The invention also relates to a process for the production of apharmaceutical according to the invention, which comprises bringing atleast one compound of the formula I into a suitable administration formusing a pharmaceutically suitable and physiologically tolerableexcipient and, if appropriate, further suitable active compounds,additives, or auxiliaries.

Suitable solid or liquid pharmaceutical preparation forms are, forexample, granules, powders, coated tablets, tablets, (micro)capsules,suppositories, syrups, juices, suspensions, emulsions, drops, orinjectable solutions, and preparations having protracted release ofactive compound, in whose preparation customary auxiliaries, such asexcipients, disintegrants, binders, coating agents, swelling agents,glidants, or lubricants, flavorings, sweeteners, and solubilizers areused. Frequently used auxiliaries which may be mentioned are, forexample, talc, starch, magnesium carbonate, titanium dioxide, lactose,mannitol and other sugars, lactoprotein, gelatin, cellulose and itsderivatives, animal and vegetable oils such as cod liver oil, sunflower,groundnut, or sesame oil, polyethylene glycols, and solvents, such as,for example, sterile water and mono- or polyhydric alcohols, e.g.,glycerol.

The pharmaceutical preparations are preferably prepared and administeredin dose units, each unit containing as active constituent a certain doseof the compound of the formula I according to the invention. In the caseof solid dose units such as tablets, capsules, coated tablets, orsuppositories, this dose can be up to approximately 1000 mg, butpreferably approximately 50 to 300 mg, and in the case of injectionsolutions in ampule form up to approximately 300 mg, but preferablyapproximately 10 to 100 mg. For the treatment of an adult patientapproximately 70 kg in weight, depending on the efficacy of the compoundof the formula I, in the case of humans and animals, daily doses ofapproximately 50 to 3000 mg of active compound are preferred. In thecase of oral administration, approximately 150 to 1000 mg is preferredand, in the case of intravenous administration, approximately 50 to 1000mg is preferred, more preferably, approximately 100 to 300 mg. Undercertain circumstances, however, higher or lower daily doses may also beappropriate. The daily dose can be administered either by singleadministration in the form of an individual dose unit or else of severalsmaller dose units or by multiple administration of subdivided doses atspecific intervals.

The invention further relates to a compound of the formula I which isoptionally coupled to polymers or solids via bridging members. These arecompounds of the formula IV ##STR9## where R¹ is the radical of theformula II or III ##STR10## L is a bridging member from the group a)--O--,

b) --NR⁵ --, in which R⁵ is a hydrogen atom,

c) --O--(CH₂)_(n) --CH₂ --, in which n is the integer 1, 2, or 3,##STR11##

in which

R³ is

a) a covalent bond or

b) --NH-- and

R⁴ is

a) a hydrogen atom or

b) a radical of an amino acid, or

e) a bridging member L, defined as under a) to d), which has a spacer,in which the spacer is a radical of the group

1) --NH--(CH₂)_(m) --S--, in which m is an integer from 1 to 12, and

X is a polymer or solid.

The radical "L-X" in the formula IV can be in the meta-, ortho-, orpara-position relative to the "NH" group on the phenyl ring, preferablyin the para-position. The term "polymer" is understood as meaning, forexample, synthetic or natural polymers from the group consisting ofpolystyrene, polypropylene, polyvinyl chloride, latex, polysaccharides,Sepharose, proteins, lipids, silicates, or nucleic acids. The polymersoptionally have to be provided with one or more functional radicals fromthe group consisting of --OH, --COOH, --NH₂ and --CO-- in order thatthey can be coupled to the compounds of the formula I. General methodsfor the coupling of the compounds of the formula I to solids aredescribed, for example, in BI Application Handbook, Ed. 1994, FIG. 4.1,(Merck AB, Uppsala, Sweden). The term "solid" represents insolublebodies which can be particulates or can occur in geometric embodimentssuch as tubes, beads, or microtiter plates.

Preferred compounds of the formula IV are those where a compound of theformula I is coupled to a solid or a polymer. Particularly preferredcompounds of the formula IV are those where a compound of the formula Isuch as 5-methylisoxazole-4-carboxylic acid(4-(2-bromoacetylamino)phenyl)amide or2-cyano-3-hydroxybut-2-enecarboxylic acid(4-(2-bromoacetylamino)phenyl)amide is coupled to a solid or a polymer.

The compounds of the formula IV are also suitable for the discovery ofspecific binding proteins from cell extracts, serum, blood, or synovialfluids, for the purification of proteins, for the modification ofmicrotiter plates or for the preparation of chromatography material, inparticular of affinity chromatography material. The proteins suitablefor purification are in direct interaction with the compounds of theformula I bound to the polymer or solid. The compounds are also suitablefor use in diagnostics.

A particularly suitable solid in the case of the compounds of theformula IV is a BlAcores® CM5 chip or a stationary phase forchromatographic investigations or separations, and also microtiterplates.

EXAMPLE 1 5-Methylisoxazole-4-carboxylic acid(4-(2-bromoacetylamino)phenyl)amide

Stage a) 5-Methylisoxazole-4-carboxylic acid (4-aminophenyl)amide

10.1 g (0.08 mol) of 5-methylisoxazole-4-carboxylic acid and 8.65 g(0.08 mol) of p-phenylenediamine are dissolved in 300 ml oftetrahydrofuran and 18.05 g (0.088 mol) of dicyclohexyicarbodiimide areadded. After 5 hours ("h"), the deposited precipitate is filtered offwith suction, the organic phase is concentrated and the product ischromatographed on silica gel by means of ethyl acetate/petroleum etherwith addition of 1% glacial acetic acid and then crystallized from ethylacetate/petroleum ether. The yield of the process was 6.8 g of acetatesalt with a melting point of 123° C. to 128° C.

Stage b) 5-Methylisoxazole-4-carboxylic acid(4-(2-bromoacetylamino)phenyl)amide

2.17 g (0.01 mol) of the product from stage a) are initially introducedinto 50 ml of tetrahydrofuran together with 1.9 g (0.016 mol) ofN-ethylmorpholine and, in an ice bath, a solution of 2.4 g (0.012 mol)of bromoacetyl bromide is added dropwise and the mixture is thenadditionally stirred at room temperature for 5 h. After addition of 5 mlof water, it is acidified to pH 2 using 1N HCI, the product is extractedwith ethyl acetate, and the organic phase is washed with water, driedand concentrated. The product is crystallized from ethylacetate/petroleum ether. The yield of the process was 2.0 g with amelting point of 179° C.

The resulting product has an ¹ H-NMR: (DMSO-d₆): 2.7 (s, 3H), 4.05 (s,2H), 7.5-7.75 (m, 4H), 9.1 (s, 1H), 10.1 and 10,45 (in each case sb,1H).

EXAMPLE 2 2-Cyano-3-hydroxybut-2-enecarboxylic acid(4-(2-bromoacetylamino)phenyl)amide

0.5 g (0.0015 mol) of the product from Example 1 is dissolved in 10 mlof tetrahydrofuran and 2 ml of 1N aqueous NaOH is added withice-cooling. The reaction is monitored by TLC and after completion(approximately after 60-90 min) the product is precipitated byacidification with 2.25 ml of 1N aqueous hydrochloric acid and additionof 100 ml of water, filtered, washed with water, then washed with alittle ethyl acetate with stirring and dried under reduced pressure. Theyield of the process was 0.28 g with a melting point greater than 210°C.

The resulting product has an ¹ H-NMR: (DMSO-d₆): 2.28 (s, 3H), 4.04 (s,2H), 7.4-7.7 (m, 4H), 8.5-10 (sb, 1H), 10.4 (sb, 2H).

EXAMPLE 3 5-Methylisoxazole-4-carboxylic acid(4-(2-aminoacetylamino)phenyl)amide-hydrochloride

Stage a) 2-tertiary-Butoxycarbonylaminoacetylamino-p-phenylenediamine

8.65 g (0.08 mol) of p-phenylenediamine are dissolved in 300 ml ofabsolute tetrahydrofuran (THF) together with 15.3 g (0.088 mol) ofN-Boc-glycine, and 18.05 g (0.088 mol) of dicyclohexylcarbodiimide isadded in portions. After 5 h, the deposited precipitate is filtered offwith suction, the filtrate is concentrated, and the product is purifiedby chromatography on silica gel by means of ethylacetate/methanol/acetic acid and then crystallized from ethylacetate/petroleum ether. The yield of the process was 13.5 g with amelting point 144° C.

Stage b) 5-Methylisoxazole-4-carbonyl chloride

127.1 g (1.0 mol) of 5-methylisoxazole-4-carboxylic acid are initiallyintroduced into 1 I of toluene, 129.8 g (1.1 mol) of thionyl chlorideare added dropwise and the mixture is then heated at 80° C. for 6 h. Thevolume is concentrated under reduced pressure to approximately one halfand the toluene solution is used directly for further reactions.

Stage c) 5-Methylisoxazole-4-carboxylic acid(4-(2-tertiarybutoxycarbonyl aminoacetylamino)phenyl)amide

13.5 g (0.05 mol) of the product from stage a) are initially introducedinto 100 ml of THF together with 7.6 ml (0.06 mol) of N-ethylmorpholineand 30 ml of the solution from stage b) (corresponds to 0.06 mol) areadded dropwise at 0° C. The mixture is stirred at room temperature for afurther 5 h, hydrolyzed with aqueous citric acid, and the product isextracted with ethyl acetate, washed with water, dried over sodiumsulfate and concentrated under reduced pressure. The yield of theprocess was 12 g with a melting point of 139° C.

Stage d) 5-Methylisoxazole4-carboxylic acid(4-(2-amino-acetylamino)phenyl)amide hydrochloride

6 g (0.017 mol) of the product from stage c) are dissolved in 180 ml ofdichloromethane and 18 ml of trifluoroacetic acid are added. The mixtureis stirred at room temperature for 1 h and concentrated, and the productis converted into the hydrochloride by dissolving it in ethanol, addingan excess of ethanolic HCl, concentrating and washing the residue bystirring with ethyl acetate. The yield of the process was 2.5 g with amelting point of 210° C.

The resulting product has an ¹ H-NMR: (DMSO-d₆): 2.7 (s, 3H), 3.7-3.9(m, 2H), 7.6 and 7.72 (in each case 2H, AA'BB'), 8.25 (sb, 2H), 9.3,10.2 and 10.75 (in each case s, 1H)

EXAMPLE 4 2-Cyano-3-hydroxybut-2-enecarboxylic acid(4-(2-aminoacetylamino)phenyl)amide trifluoroacetate

6 g (0.017 mol) of the product from Example 3, stage c) are dissolved in20 ml of 1N sodium hydroxide solution/2 ml ethanol and the mixture isstirred at room temperature until ring opening is complete(approximately 3 h). On acidifying with aqueous citric acid, theBoc-protected product is obtained in solid form and is filtered on asuction filter and dried (melting point 189° C., yield 5.5 g). 5 g ofthis intermediate product are treated with trifluoroacetic acid indichloromethane analogously to Example 3d) and the product iscrystallized from ethanol as the trifluoroacetate by means of ethylacetate and petroleum ether. The yield of the process was 5.0 g with amelting point of 209° C.

The resulting product has an ¹ H-NMR (DMSO-d₆): 2.15 (s, 3H), 3.7-3.85(m, 2H), 7.49 (s, 4H), 8.1 (sb, 3H), 10.3 and 11.2 (in each case sb,1H).

EXAMPLE 5 2-Cyano-3-hydroxybut-2-enecarboxylic acid(4-(3-iodopropoxy)phenyl)amide

Stage a) 5-Methylisoxazole-4-carboxylic acid(4-(3-iodopropoxy)phenyl)amide

6.5 g (0.03 mol) of 5-methylisoxazole-4-carboxylic acid(4-hydroxyphenyl)amide are dissolved in 57 ml (0.5 mol) of1,3-diiodopropane and 26.2 g (0.19 mol) of finely ground potassiumcarbonate are added with intensive stirring. The reaction is completeafter approximately 4 h. Working up is carried out by means offiltration, concentration and chromatography on silica gel by means ofethyl acetate/petroleum ether 1:1 with addition of 1% glacial aceticacid. The yield of the process was 6.7 g.

Stage b) 2-Cyano-3-hydroxybut-2-enecarboxylic acid(4-(3-iodo-propoxy)phenyl)amide

5.5 g of the product from stage a) are treated in 200 ml of anapproximately 1N methanolic ammonia solution until ring opening iscomplete, the mixture is concentrated, the residue is taken up withdilute acetic acid and chromatographed by means of ethylacetate/petroleum ether with addition of 1% glacial acetic acid, thencrystallized from ethyl acetate/petroleum ether. The yield of theprocess was 2.3 g with a melting point of 149° C.

The resulting product has an ¹ H-NMR (DMSO-d₆): 2.1-2.4 (m, 2H and s, 3Hat 2.3 ppm), 3.4 and 4.02 (in each case t, 2H), 6.92 and 7.42 (in eachcase 2H, AA'BB'), 7.2-8.5 (ssb, 1H), 10.2 (s, 1H).

EXAMPLE 6 2-Cyano-3-hydroxybut-2-enecarboxylic acid(4-allyloxyphenyl)amide

1.5 g (0.0068 mol) of 5-methylisoxazole-4-carboxylic acid(4-hydroxyphenyl)amide are dissolved in acetone, 5.8 ml (0.068mol) ofallyl bromide and, under intensive stirring, 9.3 g (0.068 mol) of finelyground potassium carbonate are added, then the mixture is stirredovernight at room temperature. It is filtered, the filtrate isconcentrated, and the residue is taken up in water and recrystallizedfrom ethyl acetate/petroleum ether. The yield of the process was 8.6 gwith a melting point of 162° C. to 164° C.

The resulting product has an ¹ H-NMR (DMSO-D₆): 2.3 (s, 3H), 4.45-4.63(m, 2H), 5.15-5.38 (m, 2H), 5.9-6.2 (m, 1H), 6.95 and 7.42 (in each case2H, AA'BB'), 10.0 (sb, 1H), 12.5-14.5 (ssb, 1H).

Pharmacological tests

The cell culture in vitro proliferation test is used as an activity testfor the compounds of the formula I.

EXAMPLE 7 Proliferation test

Clicks/RPMI 1640 medium (50:50) with L-glutamine and without NaHCO₃ inpowder form for 10 I (Seromed, Biochrom, Berlin, FRG), is dissolved in 9I of double-distilled water and sterile-filtered into bottles eachcontaining 900 ml.

Wash medium

900 ml of base medium are buffered with 9.5 ml of 7.5% strength sodiumhydrogencarbonate solution and 5 ml of HEPES(N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) (Gibco, Eggenstein,FRG).

Use medium

900 ml of base medium plus 19 ml of NaHCO₃ solution (7.5%; 10 ml ofHEPES solution and 10 ml of L-glutamine solution (200 mM)). The mediumused for mitogen-induced lymphocyte proliferation is use medium which isenriched with 1% heat-inactivated (30 min, 56° C.) fetal calf serum(FCS).

Tumor cell medium

For keeping tumor cells and hybridoma cells, use medium containing 5%FCS is prepared.

Culture medium for cell lines

For keeping cell lines, 900 ml of use medium containing 10% FCS, 10 mlof NEA (non-essential amino acids) solution (Gibco), 10 ml of sodiumpyruvate solution (100 mM, Gibco) and 5 ml of 10⁻² M mercaptoethanol aremixed.

Isolation and processing of the spleen cells for mitogen-inducedlymphocyte proliferation

The mice are killed by cervical dislocation and the spleens are removedunder sterile conditions. The spleens are cut up on a sterile sievehaving a mesh width of 80 mesh and, using the plunger of a plasticsyringe (10 ml), carefully strained into a Petri dish containing usemedium. To remove the erythrocytes from the spleen cell suspension, themixture is incubated at room temperature for approximately 1 min, withoccasional shaking in hypotonic, 0.17M ammonium chloride solution. Theerythrocytes are lyzed in the course of this, while the vitality andreactivity of the lymphocytes is not affected. After centrifugation (7min/340 g), the lysate is discarded, and the cells are washed twice andthen taken up in the respective test medium.

Mitogen-induced lymphocyte proliferation

5×10⁵ processed spleen cells from female NMRI mice are pipetted togetherwith various mitogens and preparation (compound of the formula I) into200 μl of test medium per hollow in flat-bottom microtiter plates. Thefollowing mitogen and preparation concentrations are used:

Concanavalin A [Serva]: 0.5-0.25-0.12 μg/ml

Lipopolysaccharide [Calbiochem]: 1.0-0.5-0.1 μg/ml

Phytohemagglutinin [Gibco]: 0.5-0.25-0.12 % stock solution

Pokeweed mitogen [Gibco] compound 1 or 2: 50, 25, 10, 7.5, 5, 2.5, 1,0.5, 0.1 μmol

The positive controls are defined as the group with mitogen additionsand without preparation. The negative controls are cells in culturemedium with preparation and without mitogen additions. Each mitogenconcentration is tested four times with all preparation concentrations.After incubation at 37° C./5% CO₂ for 48 h, 25 μl/hollow oftritium-thymidine (Amersham) having an activity of 0.25 μCi/hollow(9.25×10³ Bq) is added to the cells. A further incubation under the sameconditions for a period of 16 h follows. To evaluate the test batch, thecells are harvested on filter paper by means of a cell harvester (FlowLaboratories), nonincorporated thymidine being collected in a separatewaste bottle. The filter paper is dried, punched out and added toscintillation containers together with 2 ml of scintillator (ROTISZINT22, Roth), which are then cooled at 4° C. for a further 2 h. The amountof the radioactivity incorporated by the cells is measured in abeta-counter (Packard, TriCARB-460c).

Preparation of the tumor cells and cell lines for the proliferation test

The tumor cells or cell lines used in the test are taken from the stocksolution in the logarithmic growth phase, washed twice with wash mediumand suspended in the appropriate medium.

Carrying-out and evaluation of the proliferation tests

The proliferation test is carried out in round-bottom microtiter plates.Compounds of the formula I and interleukins are dissolved in 50 μleach/hollow of the appropriate medium and the cell count (5×10⁵) isadjusted with 100 μl/hollow so that a final volume of 200 μl/hollowresults. In all tests, the values are determined four times. Cellswithout preparations and without growth factor are defined as a negativecontrol and cells without preparation with growth factor afford thevalues for the positive control. The value of the negative control issubtracted from all values determined and the difference of positivecontrol minus negative control is set at 100%.

The microliter plates are incubated at 37° C./5% CO₂ for 72 h and theproliferation rate is determined correspondingly as in mitogen-inducedlymphocyte proliferation. The cell lines were obtained from the straincollection, American Type Culture Collection ("ATCC").

                  TABLE 1                                                         ______________________________________                                        shows the concentrations at which a 50% inhibition occurs:                        Compound of the                                                                            Cell line                                                    formula I according                                                                        A20.2.J    EL 4   K562                                             to Example (μM) (μM) (μM)                                          ______________________________________                                        1            0.8        0.8    50                                               2 15.0 6.0 20                                                                 5 50.0 25.0 not determined                                                  ______________________________________                                    

EXAMPLE 8 Process for the binding of the compound according to Example 2to the BIAcore® CM5 matrix

The starting material is a CM5 chip from Pharmacia Biosensor having acarboxymethyldextran surface (BIApplication Handbook, Ed. 1994, MerckAB, Uppsala, Sweden). The coupling steps are all carried out at a flowrate of 5 μl/min. All steps are carried out at 25° C. using Hepesbuffered saline ("HBS") pH 7.4 as the running buffer.

1st step: 35 μl of a 1:1 mixture of 0.05M NHS (N-hydroxysuccinimide) and0.2 M EDC (N-ethyl-N'-(dimethylaminopropyl)carbodiimide) in order toactivate the chip surface.

2nd step: 35 μl of a 40 mM cystamine dihydrochloride solution in 0.1Msodium borate buffer pH 8.5.

3rd step: 35 μl of ethanolamine hydrochloride pH 8.5 to saturateunoccupied matrix structures.

4th step: 35 μl of 0.1M dithiothreitol in 0.1M sodium borate buffer pH8.5.

5th step: 35 μl of a solution containing compound according to Example 2(10 mg/ml) in 0.1M sodium borate buffer pH 7.5.

EXAMPLE 9

BIAcore® method used for the analyses

The analysis was carried out essentially as described in CurrentBiology, Vol. 3, No. 12, pages 913-915 (1993).

System: BIAcore® 2000 with corresponding software, Pharmacia BiosensorAB, Uppsala, Sweden

Chip: CM5 sensor chip, Pharmacia Biosensor AB

Coating: according to Example 8

Running buffer: HBS buffer, BIA certified, Pharmacia Biosensor AB

Flow rate: 10 μl/min

Injection: 5 min association phase per 50 μl of the sample to beanalyzed.

Rinsing time: 180 sec 3 min dissociation phase

Regeneration: 2×15 sec using 0.05% sodium dodecylsulfate

    ______________________________________                                        A) Binding of various serum albumins                                                  Type    Resonance Units                                               ______________________________________                                        Bovine      50                                                                  Human 294                                                                     Rat 1061                                                                      Mouse 151                                                                     Chicken 8                                                                     Donkey 480                                                                    Sheep 331                                                                   ______________________________________                                    

The term "Resonance Units" is a quantitative unit which is proportionalto the amount of protein bound.

    ______________________________________                                        B) Binding of various dehydrogenases                                              Enzyme                Resonance Units                                     ______________________________________                                        Glyceraldehyde phosphate DH (25 nM)                                                                 69                                                        Glyceraldehyde phosphate DH (50 nM) 148                                       Glyceraldehyde phosphate DH (100 nM) 297                                      Glyceraldehyde phosphate DH (250 nM) 603                                      Dihydroorotate DH (50 nM) 203                                                 Dihydroorotate DH (100 nM) 425                                                Dihydroorotate DH (250 nM) 1064                                               Pyruvate kinase (50 nM) 85                                                    Pyruvate kinase (100 nM) 189                                                  Pyruvate kinase (250 nM) 505                                                ______________________________________                                         DH represents the enzyme designation dehydrogenase                       

The relative binding strengths of the dehydrogenases investigated werecompared with one another at a defined protein concentration. At aprotein concentration of 1 μM, the binding of the dihydroorotate DH wasthe strongest, followed by lactate DH, glyceraldehyde phosphate DH andpyruvate kinase.

EXAMPLE 10 Affinity chromatography

The compound according to Example 1 is coupled to a support matrix viaits reactive bromine group. The support material employed is FRACTOGEL®EMD-SH (Merck KGaA, Darmstadt). The covalent bonding is carried outaccording to a standard protocol as specified in DE 43 10 964. The gelobtained is packed into a SUPERFOFRMANCE® (1 cm×5 cm) column (MerckKGaA) and connected to a high-pressure liquid chromatography unit(HPLC).

Isolation and characterization of the solubile cell proteins

The strain RAW 264.7 (ATCC strain collection) is cultured for 48 h untilreaching confluence and washed free of the culture medium three timeswith cold, 4° C. PBS buffer, and the cells are transferred to PBS bufferand centrifuged at 200 g for 10 min. The cell processing is carried outat 4° C. The cell pellet is resuspended in buffer A consisting of 20 mMTRIS base, 2 mM MgCl₂ ×6 H₂ O and 1 mM dithiotreitol ("DTT"). A mixtureof various protease inhibitors is added in order to prevent proteolysisby cell-endogenous proteases. The mixture is then homogenized in agilass potter. The suspension is then centrifuged at 22,000 g for 30 minat 40° C. to obtain the soluble proteins.

The supernatant is immediately reused for affinity chromatography. Analiquot of the supernatant is retained as a reference and the remainderis pumped onto the affinity column.

HPLC

An apparatus from Kontron Instruments, Milan, Italy is employed foraffinity chromatography. The system consists of the componentsAutosampier 465, HPLC pump 422 and 422S, a high-pressure mixing valveM800, a Besta motor valve and a diode array detector 440. The datarecording and analysis are carried out using the Data System 450-MT2/DADseries. The cell supernatant is diluted to a volume of 30 ml with waterand injected at a flow rate of 1 ml/min. The breakthrough is collectedand stored at 4° C., just as the fractions up to the completion of thechromatography. The following buffers are used for the gradient elutionof the proteins:

Composition of the PBS buffer:

    ______________________________________                                        NaCl            8.00 g/l                                                        KCl 0.20 g/l                                                                  KH.sub.2 PO.sub.4 0.20 g/l                                                    Na.sub.2 HPO.sub.4 × 7H.sub.2 O 2.16 g/l                              ______________________________________                                    

Composition of the buffers used in table 2:

    ______________________________________                                        Buffer: Composition:                                                          ______________________________________                                        1       PBS                                                                     2 PBS + 0.5 M NaCl                                                            3 PBS + 150 mM A 77 1726b (sodium salt of N-(4-                                trifluoromethylphenyl)-2-cyano-3-hydroxycrotonamide)                         4 H.sub.2 O                                                                   5 6 M urea                                                                    6 60% acetonitrile + 0.1% trifluoroacetic acid (TFA)                        ______________________________________                                    

After injecting the protein solution, the gradient according to Table 2is started at a flow rate of 3 ml/min.

                  TABLE 2                                                         ______________________________________                                        Time [min] Gradient           Fraction                                        ______________________________________                                         0-10      H.sub.2 O (4)      1                                                 10-15 Gradient to PBS (1) 2                                                   15-20 PBS (1)                                                                 20-35 Gradient to PBS + 0.5 M NaCl (2) 3                                      35-40 PBS + 0.5 M NaCl (2)                                                    40-50 Gradient to PBS (2)                                                     50-55 PBS (1) 4                                                               55-70 Gradient to PBS + preparation (3) 5                                     70-75 PBS + preparation (3)                                                   75-90 H.sub.2 O (4) 6                                                          90-100 Urea (5) 7                                                            100-110 H.sub.2 O (4) 8                                                       110-120 Acetonitrile (6) 9                                                  ______________________________________                                         The numbers in parentheses relate to the buffer used.                    

The fractions are collected, dialyzed against H₂ O at 4° C. for 48 h(exclusion limit of the dialysis membrane used 6000-8000 Da) (apart fromacetonitrile fraction) and then lyophilized.

Protein determination

After the lyophilization, the proteins are dissolved in 1 ml of 1%sodium dodecylsulfate ("SDS") and diluted to 0.5% SDS with water.Aliquots of this solution are taken and employed for the proteindetermination using the BCA test (Pierce). The proteins are evaporated(UNIVASPO 150H, UniEquip Power Heater, Martinsried, Germany) and thentaken up in a suitable volume of sample buffer, consisting of 10 g ofsucrose, 9 ml of 0.25M TRIS/1M glycine solution, 7.8 ml of 10% SDS, 2.5ml of Bromophenol Blue (0.1%), 4 ml of β-mercaptoethanol and 1.7 ml ofwater.

SDS-PAGE

For the preparation of an SDS gel having a polyacrylamide gradient of10-17%, the following solutions of A and B are prepared:

    ______________________________________                                        A                B                                                            ______________________________________                                        5 ml of acrylamide (30%, 0.5%)                                                                 8.5 ml of acrylamide (30%, 0.5%)                               3.75 ml of TRIS (3 M, pH 8.8) 3.75 ml of TRIS (3 M, pH 8.8)                   6.12 ml of water 3.275 ml of glycerol                                         0.15 ml of SDS 0.15 ml of SDS                                               ______________________________________                                    

The polymerization is started by addition of 100 μl of 10% APS and 12.5μl, of TEMED. The collecting gel is prepared from 3 ml of acrylamide(10%, 0.5%), 1.36 ml of water, 1.45 ml of 0.5M TRIS (pH 6.8), 60 μl of10% SDS, 106 μl of 10% APS and 9 μl of TEMED. For the preparation of theanalytical gels, approximately 10 μg of protein per track are loaded,for preparative gels 100 μg. The determination of the molecular weightis carried out by comparison with protein standard mixtures.

Sequencing

Protein fractions which show bands of concentrated proteins in theanalytical gel are separated on a preparative scale and the separatedproteins are immobilized by blotting on a PVDF membrane (Millipore). Theproteins are visualized by staining with Coomassie and then subjecteddirectly to Edman degradation. Proteins whose N-terminus is blocked arecarefully cut out after electrophoresis on Coomassie-stained gels andwashed with water until neutral. The small pieces of gel are pressed bytwo steel sieves lying one behind the other and having a mesh width of100 μm and 32 μm and thus homogenized. The ointment-like material isdried to a low residual moisture in the UNIVAPO 150 H (UniEquip PowerHeater, Martinsried, Germany). The proteins thus obtained are cleaved topeptides by incubation at 37° C. for 7 h with the enzyme endoproteinaseLysdC (Boehringer Mannheim) (enzyme to protein ratio approximately 1:10)in a buffer consisting of 25 mM TRIS HCl, pH 8.5 and 1 mM EDTA.

The peptides obtained are eluted twice at 37° C. for 4 h using 1 ml eachof 60% acetonitrile, 0.1% TFA. The combined eluates are evaporated inthe UNIVAPO, and the peptides obtained are separated by reversed-phasechromatography. The chromatography is carried out on a LiChroCART® 125-2SUPERSPHER® 60 column using a linear gradient (11%/min) of A: 0.1% TFAin H₂ O to B: 0.1% TFA in acetonitrile in 70 min.

The amino acid sequence of the purified peptides is carried outaccording to the Edman degradation principle on a 477 A gas-phasesequencer (Applied Biosystems). The identification of the PTH-AA iscarried out on a 120A phenylthiohydantoin amino acid analyzer (AppliedBiosystems) at 269 nm.

Western Blot analysis

After the transfer of proteins from an SDS gel to the PVDF membrane, theuncoated area is blocked by incubation for 2 h with 2.5% chicken albumin(Sigma) in TBS, consisting of 45 g of NaCl, 30.3 g of TRIS base in 5 I,pH 7.4.

The first antibody is diluted in a suitable ratio with blocking bufferand incubated for 1 h. The blot is then washed three times for 5 minwith TBS and 0.1% TWEEN 20 and incubated for 1 h with the second,POD-labeled antibody. The washing process is repeated and the boundantibody is visualized by means of a solution of 60 mg of4-chloro-1-naphthol, 20 ml of methanol, 100 ml of TBS and 200 μl ofhydrogen peroxide (30%).

It was possible to identify the proteins which were obtained in the NaCl(fraction 3) and A771726b (fraction 5) as set forth in Table 2, by meansof 7 to 14 amino acid-long peptides. In the case of the 22 kDa protein,the determined amino acid sequence could be assigned to two proteins,MSP23 and NKEF-A. The reason for this lies in the high sequence homologyof the two proteins of 93%. The sequencing results of the affinitychromatography were summarized in Table 3.

                  TABLE 3                                                         ______________________________________                                                                            MW                                          Fraction Protein Amino acid sequence [Da]                                   ______________________________________                                        NaCl  Lactate DH    SADTLWGIQK      36498                                       (3) M chain (LDH-A)                                                            Cyclophilin A TAENFRALSTGEK 17971                                            A 77 HSP90-β (HSP84) EQVANSAFVERVRK 83185                                1726 HSP70 (HSC73) EIAEAYLGK 70871                                            B (5) Pyruvate kinase M.sub.2 GPEIRTGLIK 57755                                 EF1-α STTTGHLIYK 50164                                                  Acbin (y-chain) EITALAPSTMKRGILTLK 41876                                      GAPDH VIPELNGK 35679                                                          Malate DH ITPFEEKVVE-FV 35596                                                 Lactate DH NLRRVHP 36367                                                      Phosphoglycerate- PMQFLGDEETVRK 28635                                         mutase                                                                        MSP23/NKEF-A ATAVMPDGQFK 22176                                             ______________________________________                                         In the first column, the fraction number according to Table 2 is in           parentheses.                                                             

Description of the identified proteins

The 18 kDa protein concentrated in fraction (3) was identified asCyclophilin A. Cyclophilin A belongs to the large family ofpeptidylpropyl cis/trans isomerases which are involved in the mechanismof action of a number of immunosuppressant preparations, e.g.,Cyclosporin A.

Lactate dehydrogenase, an enzyme of anaerobic glycolysis which catalyzesthe reaction of pyruvate to lactate, was additionally identified infraction (3). Lactate dehydrogenase occurs in animal tissue in at leastfive different isoenzyme forms. The isoenzyme prevalent in skeletalmuscle contains four A chains, that dominant in the heart four B chains.In the present case, a peptide from the A chain of lactate dehydrogenasewas identified.

Two proteins which belong to the heat shock proteins family wereidentified in fraction (5). The heat shock protein HSP70 dominant ineukaryotes is a member of a multigene family. The heat shock proteinHSP90 is a cytosolic protein which is constitutively expressed evenunder normal conditions. It consists of two separate gene products,HSP84 and HSP86, which mutually have a sequence homology of 86%. It isknown of HSP90 that it forms a complex with steroid hormone receptorsand in this manner intervenes in the transcription of certain genes.

In fraction (5), three proteins of glycolysis were additionally foundwith glyceraldehyde-3-phosphate dehydrogenase ("GAPDH"), pyruvate kinaseM₂ and phosphoglycerate mutase. These three enzymes derive from thelower phase of glycolysis and are present under native conditions in amultienzyme complex.

A band which was also concentrated very strongly in fraction (5) wasidentified as the elongation factor ("EF-1α"). EF-1α is an importanteukaryotic translation factor. It is comparable in its function with theprokaryotic elongation factor EF-Tu and during protein biosynthesis in aGTP-dependent process transports aminoacyl-tRNAs from the cytosol totheir acceptor sites on the ribosome.

It was additionally possible to detect actin in fraction (5). Actin is aprotein occurring almost everywhere and in large amounts in eukaryotes.It is the main constituent of the musculature and of the cytoskeleton.The actin multigene family codes for at least four muscle actin formsand also for two cytoplasmic actin forms (α- and γ-actin). The actinpresent here is the γ-chain of the cytoplasmic actin form.

An enzyme of the citric acid cycle and of the malate-aspartate shuttle,malate dehydrogenase, was also identified. Malate dehydrogenase occursin animal tissue in a cytosolic and a mitochondrial isoform. Bothisoenzymes, in cooperation with aspartate aminotransferase, play animportant part in the malate-aspartate shuttle between cytosol andmitochondrium.

The 22 kDa protein of fraction (5) was assigned to two proteins on thebasis of the sequenced peptide fragment: the macrophage 23 kDa stressprotein ("MSP23") also known as osteoblast-specific factor 3 ("OSF-3")and the "natural killer cell enhancing factor-A" ("NKEF-A"). Activatedmacrophages produce reactive oxygen compounds such as H₂ O₂ and O₂.Since they themselves withstand this oxidative stress, they must have aneffective defense system in order to be able to protect themselves fromthese reactive compounds. NKEF is a cytosolic protein from human redblood cells which increases the activity of the natural killer cells.These are lymphocytes and, after cytokine stimulation, are able torecognize and to destroy a large number of tumor cells. The "naturalkiller cell enhancing factor" has a molecular weight of 44 kDa andconsists of two subunits of equal size (NKEF-A and NKEF-B), whichmutually have a good sequence homology of 88%.

EXAMPLE 11 Comparative affinity chromatography of unstimulated,LPS-stimulated and LPS-stimulated, leflunomide-treated RAW 264.7 cytosolextracts

The stimulation of the macrophage cell line RAW 264.7 withlipopolysaccharide was used as an in vitro model for inflammatoryprocesses. Lipopolysaccharide ("LPS") is an essential structuralconstituent of the outer membrane of Gram-negative bacteria and isrecognized as such by immune cells of almost all organisms. Inparticular, macrophages are activated by LPS stimulation to synthesize anumber of cytokines such as TNF-α, IL-1 and lL-6. Potential changes inthe protein pattern which accompany this stimulation should be preventedby the simultaneous administration of the immunoregulatory preparationleflunomide.

RAW 264.7 cells were incubated with 10 ng of LPS/ml of culture mediumfor 24 hours. In the case of LPS-stimulated, leflunomide-treated cells,incubation was simultaneously carried out with 60 μM A 77 1726B for 24hours. The cells were processed and the cytosolic extracts investigatedby affinity chromatography with the aid of the FRACTOGEL® columnderivatized according to Example 10. The preparation fractions of thesethree batches were then applied to an analytical gradient gel andcompared with one another.

The increase in a band around 35 kDa, which had already been identifiedby sequencing as malate dehydrogenase, was particularly clearly defined.In comparison with the other protein bands, which tended to be weaker intheir intensity on incubation with LPS and A 77 1726B, the intensity ofthese bands markedly increased.

What ds claimed is:
 1. A compound of the formula I ##STR12## or aphysiologically toierable salt of the compound of the formula I or astereoisomeric form of the compound of the formula I,where R¹ is thegroup of the formula II or III ##STR13## and wherein R² is a)--O--(CH₂)_(n) --CH═CH₂, in which n is the integer 1, 2, or 3, b) thegroup of the formula V ##STR14## in which R³ is1) halogen or 2) NH₂ andR⁴ is 1) a hydrogen atom or 2) a group of an amino acid, or c) --NH₂. 2.A compound of the formula I as claimed in claim 1, where R¹ is the,group of the formula II or III and wherein R² isa) --O--CH₂ -CH═CH₂, orb) --NH--C(O)--CH(R³)(R⁴), in which R³ is bromine, --NH₂, or chlorineand R⁴ is a hydrogen atom.
 3. A compound of the formula I as claimed inclaim 1, wherein the group R² in formula I is in the meta-, ortho-, orpara-position relative to the "NH" group on the phenyl ring.
 4. Acompound as claimed in claim 3, wherein the group R² in formula I is inthe para-position relative to the "NH" group on the phenyl ring.
 5. Acompound as claimed in claim 2, wherein the group R² in formula I is inthe meta-, ortho-, or para-position relative to the "NH" group on thephenyl ring.
 6. A compound as claimed in claim 5, wherein the group R²in formula I is in the para-position relative to the "NH" group on thephenyl ring.
 7. A compound having a formula2-cyano-3-hydroxybut-2-enecarboxylic acid (4-allyloxyphenyl)amide,2-cyano-3-hydroxybut-2-enecarboxylic acid(4-(2-amino-acetylamino)phenyl)-amide trifluoroacetate,5-methylisoxazole-4-carboxylic acid (4-(2-aminoacetylamino)-phenyl)amidehydrochloride, 2-cyano-3-hydroxybut-2-enecarboxylic acid(4-(2-bromoacetylamino) phenyl)amide, or 5-methylisoxazole-4-carboxylicacid (4-(2-bromoacetylamino)-phenyl)amide, or a physiologicallytolerable salt of any of the foregoing compounds.
 8. A pharmaceuticalcomposition comprising a compound of the formula I ##STR15## or aphysiologically tolerable salt of the compound of the formula I or astereoisomeric form of the compound of the formula I, where R¹ is thegroup of the formula II or Ill ##STR16## and wherein R² is a)--O--(CH₂)_(n) --CH═CH₂, in which n is the integer 1, 2, or 3,b) thegroup of the formula V ##STR17## in which R³ is1) halogen or 2) NH₂ andR⁴ is1) a hydrogen atom or 2) a group of an amino acid, or c) --NH₂,anda pharmaceutically tolerable excipient.
 9. A method for the treatmentof a lung cancer, leukemia, Kaposi's sarcoma, ovarian cancer, sarcoma,meningioma, intestinal cancer, cancer of the lymph nodes, brain tumors,breast cancer, stomach cancer, cancer of the pancreas, cancer of theprostrate, skin cancer an inflammation, or systematic lupuserythematosus ("SLE"), type II diabetes, myasthenia gravis, Sjogrensyndrome, dermatomyositis, sclerodermatitis, multiple sclerosis ("MS")comprising administering an effective amount of a compound of theformula I ##STR18## or a physiologically tolerable salt of the compoundof the formula I or a stereoisomeric form of the compound of the formulaI, where R¹ is the group of the formula II or III ##STR19## and whereinR² is a) --O--(CH₂)_(n) --CH═CH₂, in which n is the integer 1, 2, or3,b) the group of the formula V ##STR20## in which R³ is1) halogen or 2)NH₂ and R⁴ is1) a hydrogen atom or 2) a group of an amino acid, or c)--NH₂.
 10. A method for the treatment of a lung cancer, leukemia,Kaposi's sarcoma, ovarian cancer, sarcoma, meningioma, intestinalcancer, cancer of the lymph nodes, brain tumors, breast cancer, stomachcancer, cancer of the pancreas, cancer of the prostrate, skin cancer aninflammation, or systematic lupus erythematosus ("SLE"), type IIdiabetes, myasthenia gravis, Sjogren syndrome, dermatomyositis,sclerodermatitis, multiple sclerosis ("MS") comprising administering aneffective amount of a pharmaceutical composition as claimed in claim 8.